RNA Oligonucleotides Research Articles

Monitoring Real-Time Temperature Dynamics of a Short RNA Hairpin Using Förster Resonance Energy Transfer and Circular Dichroism.

RNA molecules play crucial roles in gene expression regulation and cellular signaling, and these functions are governed by the formation of RNA secondary and tertiary structures. These structures are highly dynamic and subject to rapid changes in response to environmental cues, temperature in particular. Thermosensitive RNA secondary structures have been harnessed by multiple organisms to survey their temperature environment and to adjust gene expression accordingly. It is thus highly desirable to observe RNA structural changes in real time over a range of temperatures. Multiple approaches have been developed to study structural dynamics, but many of these require extensive processing of the RNA, large amounts of RNA input, and/or cannot be applied under physiological conditions. Here, we describe the use of a dually fluorescently labeled RNA oligonucleotide (containing a predicted hairpin structure) to monitor subtle RNA structural dynamics in vitro by Förster resonance energy transfer (FRET) and circular dichroism (CD) spectroscopy. These approaches can be employed under physiologically relevant conditions over a range of temperatures and with RNA concentrations as low as 200nM; they enable us to observe RNA structural dynamics in real time and to correlate these dynamics with changes in biological processes such as translation.

Cell-SELEX and application research of a DNA aptamer against esophageal squamous cell carcinoma (ESCC) cell line TE-1.

Esophageal cancer is a common cancer with high morbidity and mortality that severely threatens the safety and quality of human life. The strong metastatic nature of esophageal cancer enables it to metastasize more quickly and covertly, making it difficult for current diagnostic and treatment methods to achieve efficient early screening, as well as timely and effective treatment. As a promising solution, nucleic acid aptamers, a kind of special single-stranded DNA or RNA oligonucleotide selected by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology, can specifically bind with different molecular targets. In this paper, random DNA single-stranded oligonucleotides were used as the initial library. Using TE-1 cells and HEEC cells as targets, specific binding sequences were selected by 15 rounds of the cell-SELEX method, and the aptamer sequence that binds to TE-1 cells with the most specificity was obtained and named Te4. The Te4 aptamer was further validated for binding specificity, binding affinity, type of target, in vitro cytotoxicity when conjugated with DOX(Te4-DOX), and in vivo distribution. Results of in vitro validation showed that Te4 has outstanding binding specificity with a Kd value of 51.16 ± 5.52 nM, and the target type of Te4 was preliminarily identified as a membrane protein. Furthermore, the cytotoxicity experiment showed that Te4-DOX has specific cytotoxicity towards cultured TE-1 cells. Finally, the results of the in vivo distribution experiment showed that the Te4 aptamer is able to specifically target tumor regions in nude mice, showing great potential to be applied in future diagnosis and targeted therapy of esophageal cancer.

Circular RNA oligonucleotides: enzymatic synthesis and scaffolding for nanoconstruction.

We report the efficient synthesis of monomeric circular RNAs (circRNAs) in the size range of 16-44 nt with a novel DNA dumbbell splinting plus T4 DNA ligation strategy. Such a DNA dumbbell splinting strategy was developed by one group among ours recently for near-quantitative conversion of short linear DNAs into monomeric circular ones. Furthermore, using the 44 nt circRNA as scaffold strands, we constructed hybrid RNA:DNA and pure RNA:RNA double crossover tiles and their assemblies of nucleic acid nanotubes and flat arrays.

Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking Agent.

Due to their unique three-dimensional structure, DNA or RNA oligonucleotide aptamers bind to various molecules with high affinity and specificity. Aptamers, alone or in combination with antibodies, can be used to sensitively quantify target molecules by quantitative real-time polymerase chain reaction (qPCR). However, the assays are often complicated and unreliable. In this study, we explored the feasibility of performing the entire assay on wells of routinely used polypropylene PCR plates. We found that polypropylene wells efficiently bind proteins. This allows the entire assay to be run in a single well. To minimize nonspecific binding of the assay components to the polypropylene wells, we tested various blocking agents and identified methylcellulose as an effective alternative to the commonly used BSA. Methylcellulose not only demonstrates comparable or superior blocking capabilities but also offers the advantage of a well-defined composition and non-animal origin. Our findings support the utilization of aptamers, either alone or in combination with antibodies, for sensitive quantification of selected molecules immobilized in polypropylene PCR wells in a streamlined one-well qPCR assay under well-defined conditions.

APIPred: An XGBoost-Based Method for Predicting Aptamer-Protein Interactions.

Aptamers are single-stranded DNA or RNA oligos that can bind to a variety of targets with high specificity and selectivity and thus are widely used in the field of biosensing and disease therapies. Aptamers are generated by SELEX, which is a time-consuming procedure. In this study, using in silico and computational tools, we attempt to predict whether an aptamer can interact with a specific protein target. We present multiple data representations of protein and aptamer pairs and multiple machine-learning-based models to predict aptamer-protein interactions with a fair degree of selectivity. One of our models showed 96.5% accuracy and 97% precision, which are significantly better than those of the previously reported models. Additionally, we used molecular docking and SPR binding assays for two aptamers and the predicted targets as examples to exhibit the robustness of the APIPred algorithm. This reported model can be used for the high throughput screening of aptamer-protein pairs for targeting cancer and rapidly evolving viral epidemics.

RNA G-quadruplexes inhibit translation of the PE/PPE transcripts in Mycobacterium tuberculosis

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures invitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M.smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies.

Cyclophospholipids Enable a Protocellular Life Cycle.

There is currently no plausible path for the emergence of a self-replicating protocell, because prevalent formulations of model protocells are built with fatty acid vesicles that cannot withstand the concentrations of Mg2+ needed for the function and replication of nucleic acids. Although prebiotic chelates increase the survivability of fatty acid vesicles, the resulting model protocells are incapable of growth and division. Here, we show that protocells made of mixtures of cyclophospholipids and fatty acids can grow and divide in the presence of Mg2+-citrate. Importantly, these protocells retain encapsulated nucleic acids during growth and division, can acquire nucleotides from their surroundings, and are compatible with the nonenzymatic extension of an RNA oligonucleotide, chemistry needed for the replication of a primitive genome. Our work shows that prebiotically plausible mixtures of lipids form protocells that are active under the conditions necessary for the emergence of Darwinian evolution.

Antiviral activity of porphyrins and porphyrin-like compounds against tick-borne encephalitis virus: Blockage of the viral entry/fusion machinery by photosensitization-mediated destruction of the viral envelope

Tick-borne encephalitis virus (TBEV), the causative agent of tick-borne encephalitis (TBE), is a medically important flavivirus endemic to the European–Asian continent. Although more than 12,000 clinical cases are reported annually worldwide, there is no anti-TBEV therapy available to treat patients with TBE. Porphyrins are macrocyclic molecules consisting of a planar tetrapyrrolic ring that can coordinate a metal cation. In this study, we investigated the cytotoxicity and anti-TBEV activity of a large series of alkyl- or (het)aryl-substituted porphyrins, metalloporphyrins, and chlorins and characterized their molecular interactions with the viral envelope in detail. Our structure–activity relationship study showed that the tetrapyrrole ring is an essential structural element for anti-TBEV activity, but that the presence of different structurally distinct side chains with different lengths, charges, and rigidity or metal cation coordination can significantly alter the antiviral potency of porphyrin scaffolds. Porphyrins were demonstrated to interact with the TBEV lipid membrane and envelope protein E, disrupt the TBEV envelope and inhibit the TBEV entry/fusion machinery. The crucial mechanism of the anti-TBEV activity of porphyrins is based on photosensitization and the formation of highly reactive singlet oxygen. In addition to blocking viral entry and fusion, porphyrins were also observed to interact with RNA oligonucleotides derived from TBEV genomic RNA, indicating that these compounds could target multiple viral/cellular structures. Furthermore, immunization of mice with porphyrin-inactivated TBEV resulted in the formation of TBEV-neutralizing antibodies and protected the mice from TBEV infection. Porphyrins can thus be used to inactivate TBEV while retaining the immunogenic properties of the virus and could be useful for producing new inactivated TBEV vaccines.

Impact of the Core Chemistry of Self‐Assembled Spherical Nucleic Acids on their In Vitro Fate

AbstractNucleic acid therapeutics (NATs), such as mRNA, small interfering RNA or antisense oligonucleotides are extremely efficient tools to modulate gene expression and tackle otherwise undruggable diseases. Spherical nucleic acids (SNAs) can efficiently deliver small NATs to cells while protecting their payload from nucleases, and have improved biodistribution and muted immune activation. Self‐assembled SNAs have emerged as nanostructures made from a single DNA‐polymer conjugate with similar favorable properties as well as small molecule encapsulation. However, because they maintain their structure by non‐covalent interactions, they might suffer from disassembly in biologically relevant conditions, especially with regard to their interaction with serum proteins. Here, we report a systematic study of the factors that govern the fate of self‐assembled SNAs. Varying the core chemistry and using stimuli‐responsive disulfide crosslinking, we show that extracellular stability upon binding with serum proteins is important for recognition by membrane receptors, triggering cellular uptake. At the same time, intracellular dissociation is required for efficient therapeutic release. Disulfide‐crosslinked SNAs combine these two properties and result in efficient and non‐toxic unaided gene silencing therapeutics. We anticipate these investigations will help the translation of promising self‐assembled structures towards in vivo gene silencing applications.

Impact of the Core Chemistry of Self-Assembled Spherical Nucleic Acids on their In Vitro Fate.

Nucleic acid therapeutics (NATs), such as mRNA, small interfering RNA or antisense oligonucleotides are extremely efficient tools to modulate gene expression and tackle otherwise undruggable diseases. Spherical nucleic acids (SNAs) can efficiently deliver small NATs to cells while protecting their payload from nucleases, and have improved biodistribution and muted immune activation. Self-assembled SNAs have emerged as nanostructures made from a single DNA-polymer conjugate with similar favorable properties as well as small molecule encapsulation. However, because they maintain their structure by non-covalent interactions, they might suffer from disassembly in biologically relevant conditions, especially with regard to their interaction with serum proteins. Here, we report a systematic study of the factors that govern the fate of self-assembled SNAs. Varying the core chemistry and using stimuli-responsive disulfide crosslinking, we show that extracellular stability upon binding with serum proteins is important for recognition by membrane receptors, triggering cellular uptake. At the same time, intracellular dissociation is required for efficient therapeutic release. Disulfide-crosslinked SNAs combine these two properties and result in efficient and non-toxic unaided gene silencing therapeutics. We anticipate these investigations will help the translation of promising self-assembled structures towards in vivo gene silencing applications.

Abstract 12705: Identification and Characterization of Solbinsiran, a GalNAc Conjugated siRNA Targeting Angiopoietin-Like 3

Introduction: While statins play an important role in reducing LDL-C levels and the risk of ASCVD events, residual risk from apolipoprotein B (apoB)-containing lipoprotein particles persists among patients with or at high risk of ASCVD. Hypothesis: Blocking ANGPTL3 activity or preventing its expression is a therapeutic approach to reduce apoB-containing lipoprotein particles. Solbinsiran (LY3561774), a novel Dicer-substrate small interfering RNA oligonucleotide, was designed to reduce the levels of ANGPTL3 protein expression. Methods: Nucleotide base sequences of a library of anti-ANGPTL3 siRNAs were selected using an in silico RNAi prediction algorithm on ANGPTL3 mRNA sequences conserved in humans and cynomolgus monkeys. Through a large-scale in vitro screen of the anti-ANGPTL3 library in human cells, the candidate solbinsiran sequence was identified as an efficacious siRNA. Solbinsiran in vivo efficacy was measured by RT-qPCR of liver from CD-1 female mice overexpressing human ANGPTL3 . The degree and durability of ANGPTL3 mRNA knockdown and circulating protein level reduction by solbinsiran was evaluated in cynomolgus monkeys administered single dose subcutaneous injections of either PBS or 3 mg/kg solbinsiran. Blood samples to measure ANGPTL3 protein were collected on Study Days -8, -5, and 0, and weekly after dosing until Day 113. Ultrasound-guided core needle liver biopsies were collected on Study Days 5, 28, 56, 84, and 113. Total RNA derived from the liver biopsy samples to measure ANGPTL3 mRNA was subjected to RT-qPCR analysis. Results: When given a single dose at 1 mg/kg subcutaneously, solbinsiran reduced human ANGPTL3 mRNA by 65% in hepatocytes as compared to vehicle-treated mice. A single dose of solbinsiran reduced liver ANGPTL3 mRNA expression by up to 72.9% (p<0.0001) and serum ANGPTL3 protein expression by 68.6% (p<0.001). Solbinsiran also displayed a long duration of activity, as indicated by a greater than 50% mRNA knockdown remaining 12 weeks after a single dose. Conclusions: These data supported the advancement of solbinsiran to Phase 1 clinical testing in participants with mixed dyslipidemia (ClinicalTrials.gov Identifier: NCT04644809).

Effect of montmorillonite K10 clay on RNA structure and function

One of the earliest living systems was likely based on RNA (“the RNA world”). Mineral surfaces have been postulated to be an important environment for the prebiotic chemistry of RNA. In addition to adsorbing RNA and thus potentially reducing the chance of parasitic takeover through limited diffusion, minerals have been shown to promote a range of processes related to the emergence of life, including RNA polymerization, peptide bond formation, and self-assembly of vesicles. In addition, self-cleaving ribozymes have been shown to retain activity when adsorbed to the clay mineral montmorillonite. However, simulation studies suggest that adsorption to minerals is likely to interfere with RNA folding and, thus, function. To further evaluate the plausibility of a mineral-adsorbed RNA world, here we studied the effect of the synthetic clay montmorillonite K10 on the malachite green RNA aptamer, including binding of the clay to malachite green and RNA, as well as on the formation of secondary structures in model RNA and DNA oligonucleotides. We evaluated the fluorescence of the aptamer complex, adsorption to the mineral, melting curves, Förster resonance energy transfer interactions, and 1H-NMR signals to study the folding and functionality of these nucleic acids. Our results indicate that while some base pairings are unperturbed, the overall folding and binding of the malachite green aptamer are substantially disrupted by montmorillonite. These findings suggest that minerals would constrain the structures, and possibly the functions, available to an adsorbed RNA world.

An RNA-Ligation-Based RACE-PAT Assay to Monitor Poly(A) Tail Length of mRNAs of Interest.

In eukaryotes, a non-templated poly-adenosine (poly(A)) tail is added co-transcriptionally to almost every messenger RNA (mRNA). The length of this poly(A) tail changes during the lifetime of mRNAs and has been shown in many circumstances to be an important factor controlling transcript fates. Yet, the measure of the length of this homogenous nucleotide sequence is technically challenging, making it difficult to assess its dynamic variation. In this chapter, we describe an RNA-ligation-based RACE-PAT (Rapid Amplification of cDNA End-Poly(A) Tail) assay to monitor the poly(A) tail length of mRNAs. In the first step, an RNA oligonucleotide is ligated to mRNA 3' ends providing an anchoring site to prime cDNA synthesis, avoiding the bias introduced by oligo(dT)-derived primers. Afterward, reverse transcription is performed with an anchor primer with a unique 5' extension. The choice of the oligonucleotide 3' end at this step allows further flexibility to amplify modified tails, for example, by uridylation. Next, short DNA fragments encompassing the poly(A) tails are amplified by Polymerase Chain Reaction (PCR) using as forward primer, a transcript-specific primer hybridizing close to the transcript polyadenylation signal, and as reverse primer, an oligonucleotide corresponding to the 5' extension of the primer used for cDNA synthesis, ensuring that only cDNAs are amplified. The resulting DNA fragments are then visualized after size fractionation by electrophoresis. This method does not provide exact nucleotide count and composition but has the advantage of allowing the processing of many samples in parallel at a low cost.

Bioorthogonal Aptamer-ATTEC Conjugates for Degradation of Alpha-Synuclein via Autophagy-Lysosomal Pathway.

Autophagosome-tetheringcompound (ATTEC) technology has recently been emerging as a novel approach for degrading proteins of interest (POIs). However, it still faces great challenges in how to design target-specific ATTEC molecules. Aptamers are single-stranded DNA or RNA oligonucleotides that can recognize their target proteins with high specificity and affinity. Here, ATTEC is combined with aptamers for POIs degradation. As a proof of concept, pathological protein α-synuclein (α-syn) is chosen as the target and an efficient α-syn degrader is generated. Aptamer as a targeting warhead of α-syn is conjugated with LC3B-binding compound 5,7-dihydroxy-4-phenylcoumarin (DP) via bioorthogonal click reaction. It is demonstrated that the aptamer conjugated with DP is capable of clearing α-syn through LC3 and autophagic degradation. These results indicate that aptamer-based ATTECs are a versatile approach to degrade POIs by taking advantage of the well-defined different aptamers for targeting diverse proteins, which provides a new way for the design of ATTECs to degradation of targeted proteins.

B-375 Mer-idpcr: A Method for Sequence-specific Measurement of Small RNA Expression and Methylation in Liquid Biopsies

Abstract Background Small RNAs are an important class of biomarkers in liquid biopsy for cancer diagnostics. New discoveries in the field of “epitranscriptomics” have uncovered further functional layers of tumor biology that are ignored by standard gene expression measurement methods. Therefore, there is a pressing need for sequence specific methods that can detect methylation stoichiometry on small RNAs purified from liquid biopsies in a diagnostic setting. Methods To detect small RNAs specifically, we designed structured oligonucleotide adapters that bear reverse complementary overhangs to both the 5′ and 3′ end of target RNAs. After nick-based ligation, reverse transcription under limiting dNTP conditions enables the differential processing of methylated vs unmethylated species. Finally, target specific TaqMan probes are used on a digital PCR based platform for final quantification and calculation of methylation stoichiometry. Results We show that mer-idPCR can detect target RNAs in a sequence-specific manner using synthetic cognate RNA oligos and specificity controls, such as nucleotide variants or non-target miRNAs. Detected RNA is quantified in a linear range with a limit of detection at 40 copies/µL of cDNA. Concurrently, the method can detect 2′-o-methylation in 0%–100% range with a resolution of 10%. mer-idPCR can be applied to RNA extracted from various sources including serum, plasma or whole blood stabilizing tubes. Conclusion mer-idPCR can enable the high resolution, sequence specific quantification of target small RNA species, in addition to the measurement of 2′-o-methylation site status, in small RNAs extracted from peripheral blood. We show the adapter design requirements and discuss optimizations of adapter design and reverse transcription conditions that led to improved specificity, reproducibility, and linearity of this method. mer-idPCR enables the validation and discovery of novel biomarkers for cancer detection in liquid biopsies.

Bispecific G-quadruplexes as inhibitors of cancer cells growth

A therapeutic system with the ability to target more than one protein is an important aim of cancer therapy since tumor growth is accompanied by dysregulation of many biological pathways. G-quadruplexes (G4s) are non-canonical structures formed by guanine-rich DNA or RNA oligonucleotides, with the ability to bind to different targets. In this study, we constructed ten novel bispecific G-quadruplex conjugates based on AT11, TBA, T40214 and T40231 aptamer structures, with the ability to bind two different targets at once in cancer cells. We analyzed the physicochemical aspects and the anticancer properties of novel molecules relating them with the single G-quadruplex unit and attempted to comprehend the correlation between the structures of bispecific G-quadruplexes with their biological activity. Our studies uncovered conjugates with considerable antiproliferative potential in HeLa and MCF-7 cancer cell lines, however with relatively low thermal stability or low nuclease resistance. Three conjugates among all studied oligonucleotides possess improved antiproliferative activity in MCF-7 cell line in comparison to their single G-quadruplex units leading to up to 90% inhibition of cancer cells growth, but their inhibitory potential is rather comparable to the effect observed for mix of two separate G-quadruplex units. Importantly, the conjugation enhances oligonucleotides enzymatic stability leading to the improvement of their therapeutic profile. The comprehensive studies presented herein indicate new approach for possibly effective cancer therapy and for the design of G4-based drugs.

Modulation of the tetrameric I-motif folding of C-rich Tetrahymena telomeric sequences by hexitol nucleic acid (HNA) modifications

I-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine+ base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 2′-deoxyribose, arabinose, and 2′-deoxy-2′-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications.An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 3′-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for I-motif characterization and applications.

Charge-State-Dependent Collision-Induced Dissociation Behaviors of RNA Oligonucleotides via High-Resolution Mass Spectrometry.

Mass spectrometry (MS)-based analysis of RNA oligonucleotides (oligos) plays an increasingly important role in the development of RNA therapeutics and epitranscriptomics research. However, MS fragmentation behaviors of RNA oligomers are understood insufficiently. Herein, we characterized the negative-ion-mode fragmentation behaviors of 26 synthetic RNA oligos containing four to eight nucleotides using collision-induced dissociation (CID) on a high-resolution, accurate-mass instrument. We found that in CID spectra acquired under the normalized collision energy (NCE) of 35%, approximately 70% of the total peak intensity was attributed to sequencing ions (a-B, a, b, c, d, w, x, y, z), around 25% of the peak intensity came from precursor ions that experienced complete or partial loss of a nucleobase in the form of either a neutral or an anion, and the remainder were internal ions and anionic nucleobases. The top five sequencing ions were the y, c, w, a-B, and a ions. Furthermore, we observed that CID fragmentation behaviors of RNA oligos were significantly impacted by their precursor charge. Specifically, when the precursors had a charge from 1- to 5-, the fractional intensity of sequencing ions decreased, while that of precursors that underwent either neutral or charged losses of a nucleobase increased. Additionally, we found that RNA oligos containing 3'-U tended to produce precursors with HNCO and/or NCO- losses, which presumably corresponded to isocyanic acid and cyanate anion, respectively. These findings provide valuable insights for better comprehending the mechanism behind RNA fragmentation by MS/MS, thereby facilitating the future automated identification of RNA oligos based on their CID spectra in a more efficient manner.

A ribonuclease activity linked to DYW1 in vitro is inhibited by RIP/MORF proteins

Organellar C-to-U RNA editing in plants occurs in complexes composed of various classes of nuclear-encoded proteins. DYW-deaminases are zinc metalloenzymes that catalyze hydrolytic deamination required for C-to-U modification editing. Solved crystal structures for DYW-deaminase domains display all structural features consistent with a canonical cytidine deamination mechanism. However, some recombinant DYW-deaminases from plants have been associated with ribonuclease activity in vitro. Direct ribonuclease activity by an editing factor is confounding since it is not required for deamination of cytosine, theoretically would be inimical for mRNA editing, and does not have a clear physiological function in vivo. His-tagged recombinant DYW1 from Arabidopsis thaliana (rAtDYW1) was expressed and purified using immobilized metal affinity chromatography (IMAC). Fluorescently labeled RNA oligonucleotides were incubated with recombinant AtDYW1 under different conditions. Percent relative cleavage of RNA probes was recorded at multiple time points from triplicate reactions. The effects of treatment with zinc chelators EDTA and 1, 10-phenanthroline were examined for rAtDYW1. Recombinant His-tagged RNA editing factors AtRIP2, ZmRIP9, AtRIP9, AtOZ1, AtCRR4, and AtORRM1 were expressed in E. coli and purified. Ribonuclease activity was assayed for rAtDYW1 in the presence of different editing factors. Lastly, the effects on nuclease activity in the presence of nucleotides and modified nucleosides were investigated. RNA cleavage observed in this study was linked to the recombinant editing factor rAtDYW1 in vitro. The cleavage reaction is sensitive to high concentrations of zinc chelators, indicating a role for zinc ions for activity. The addition of equal molar concentrations of recombinant RIP/MORF proteins reduced cleavage activity associated with rAtDYW1. However, addition of equal molar concentrations of purified recombinant editing complex proteins AtCRR4, AtORRM1, and AtOZ1 did not strongly inhibit ribonuclease activity on RNAs lacking an AtCRR4 cis-element. Though AtCRR4 inhibited AtDYW1 activity for oligonucleotides with a cognate cis-element. The observation that editing factors limit ribonuclease activity of rAtDYW1 in vitro, suggests that nuclease activities are limited to RNAs in absence of native editing complex partners. Purified rAtDYW1 was associated with the hydrolysis of RNA in vitro, and activity was specifically inhibited by RNA editing factors.

Sequence- and structure-specific RNA oligonucleotide binding attenuates heterogeneous nuclear ribonucleoprotein A1 dysfunction.

The RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (A1) regulates RNA metabolism, which is crucial to maintaining cellular homeostasis. A1 dysfunction mechanistically contributes to reduced cell viability and loss, but molecular mechanisms of how A1 dysfunction affects cell viability and loss, and methodologies to attenuate its dysfunction, are lacking. Utilizing in silico molecular modeling and an in vitro optogenetic system, this study examined the consequences of RNA oligonucleotide (RNAO) treatment on attenuating A1 dysfunction and its downstream cellular effects. In silico and thermal shift experiments revealed that binding of RNAOs to the RNA Recognition Motif 1 of A1 is stabilized by sequence- and structure-specific RNAO-A1 interactions. Using optogenetics to model A1 cellular dysfunction, we show that sequence- and structure-specific RNAOs significantly attenuated abnormal cytoplasmic A1 self-association kinetics and A1 cytoplasmic clustering. Downstream of A1 dysfunction, we demonstrate that A1 clustering affects the formation of stress granules, activates cell stress, and inhibits protein translation. With RNAO treatment, we show that stress granule formation is attenuated, cell stress is inhibited, and protein translation is restored. This study provides evidence that sequence- and structure-specific RNAO treatment attenuates A1 dysfunction and its downstream effects, thus allowing for the development of A1-specific therapies that attenuate A1 dysfunction and restore cellular homeostasis.